The treatment of metastatic malignancies is hindered by a poor understanding of the tumor-host interactions underlying disease dissemination. Particularly, the mechanisms by which the overall patient's well-being impacts cancer progression remain unclear. We propose a sympathetic neurotransmitter, neuropeptide Y (NPY), as a novel metastatic factor, which can be released systemically due to sympathetic system activation or secreted from tumors. Among them, two pediatric tumors, neuroblastoma (NB) and Ewing sarcoma (ES), are particularly rich in NPY. Strikingly, in both malignancies, high serum NPY in patients associates with metastases and bone involvement. Moreover, we have found that NPY stimulates motility and invasiveness of tumor cells, while expression of the peptide and its receptors is markedly enhanced in invasive and metastatic cells. NPY is particularly highly expressed in bone metastases, while NPY-rich ES cells give rise to frequent bone metastases in orthotopic xenograft model. Notably, in both NB and ES, these metastatic actions of NPY are mediated by the hypoxia-inducible, Y2R/Y5R pathway. The same receptors are also present on endothelial cells and mediate angiogenic actions of NPY. Based on this, we hypothesize that the dissemination of NB and ES is driven by the common, disease-independent actions of NPY, such as 1) an increase in tumor cell proliferation, motility and invasiveness; 2) angiogenesis; 3) promoting bone invasion by blocking osteoblast differentiation and stimulating the release of osteolytic factors. The same mechanisms can contribute to the dissemination of other tumors, without endogenous NPY expression, when systemic NPY levels are elevated due to high sympathetic activity. Thus, we postulate that that NPY axis may be successfully targeted to prevent metastasis. In the current study, we will focus on deciphering these universal processes, rather than tumor-specific features, using NB and ES as models of NPY-rich tumors. To this end, we will 1) Identify NPY-dependent stages of metastases formation; 2) Determine cellular and molecular mechanisms of NPY actions, and 3) Assess NPY receptors as potential therapeutic targets. Initially, the effect of NPY knock-down or overexpression on metastases formation will be tested using animal models. For ES, orthotopic xenografts will be subjected to hypoxia by arterial occlusion to assess NPY as a mediator of hypoxia-induced increase in tumor malignancy. Having determined NPY-dependent steps of metastases, we will identify mechanisms of its actions and mediating receptors using in vitro assays recapitulating stages of metastases. We will also determine NPY-induced changes in signaling pathway activation, gene expression and factors secreted from tumor cells. Lastly, we will test if blocking the identified receptors and pathways inhibits metastatic actions of NPY in vitro and in vivo. This study will be the first to test metastatic properties of NPY and assess its value as a target for anti- metastatic therapies. While this project will focus on tumors with high NPY expression, our data will provide a foundation for future studies on the role of systemic, sympathetic neuron-derived NPY in cancer progression.